DNA replication in genetic hereditary matrial

1. Replication
1

2. DNA RNA protein
transcription translation
replication
reverse
transcription
Central dogma
2

3. • Replication: synthesis of daughter
DNA from parental DNA
• Transcription: synthesis of RNA using
DNA as the template
• Translation: protein synthesis using
mRNA molecules as the template
• Reverse transcription: synthesis of
DNA using RNA as the template
3

4. DNA
Replication
4

5. General Concepts of
DNA Replication

6. DNA replication
• A reaction in which daughter DNAs are
synthesized using the parental DNAs as
the template.
• Transferring the genetic information to
the descendant generation with a high
fidelity
replication
parental DNA
daughter DNA
6

7. Daughter strand synthesis
• Chemical formulation:
• The nature of DNA replication is a
series of 3´- 5´phosphodiester bond
formation catalyzed by a group of
enzymes.
7

8. The DNA backbone
• Putting the DNA backbone
together
– refer to the 3 and 5 ends of
the DNA
OH
O
PO4
base
CH2
O
base
O
P
O
C
O
–O
CH2
1
2
4
5
1
2
3
3
4
5

9. Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a
free 3´-OH end
Enzyme: DNA-dependent DNA
polymerase (DDDP),
other enzymes,
protein factor
DNA replication system
9

10. Characteristics of replication
 Semi-conservative replication
 Bidirectional replication
 Semi-continuous replication
 High fidelity
10

11. §1.1 Semi-Conservative Replication
11

12. Semiconservative replication
Half of the parental DNA molecule is
conserved in each new double helix,
paired with a newly synthesized
complementary strand. This is called
semiconservative replication
12

13. Semiconservative replication
13

14. Experiment of DNA semiconservative replication
"Heavy" DNA(15
N)
grow in 14
N
medium
The first
generation
grow in 14
N
medium
The second
generation
14

15. Significance
The genetic information is ensured to be
transferred from one generation to the
next generation with a high fidelity.
15

16. §1.2 Bidirectional Replication
• Replication starts from unwinding the
dsDNA at a particular point (called
origin), followed by the synthesis on
each strand.
• The parental dsDNA and two newly
formed dsDNA form a Y-shape
structure called replication fork.
16

17. 3'
5'
5'
3'
5'
3'
5'
3'
direction of
replication
Replication fork
17

18. Bidirectional replication
• Once the dsDNA is opened at the
origin, two replication forks are
formed spontaneously.
• These two replication forks move in
opposite directions as the syntheses
continue.
18

19. Replication of prokaryotes
The replication
process starts
from the origin,
and proceeds
in two opposite
directions. It is
named 
replication.
19

20. Replication of eukaryotes
• Chromosomes of eukaryotes have
multiple origins.
• The space between two adjacent
origins is called the replicon, a
functional unit of replication.
20

21. 5'
3'
3'
5'
bidirectional replication
fusion of bubbles
3'
5'
3'
5'
5'
3'
5'
3'
origins of DNA replication (every ~150 kb)
21

22. §1.3 Semi-continuous Replication
The daughter strands on two template
strands are synthesized differently since
the replication process obeys the
principle that DNA is synthesized from
the 5´ end to the 3´end.
22

23. 5'
3'
3'
5'
5'
direction of unwinding
3'
On the template having the 3´- end, the
daughter strand is synthesized
continuously in the 5’-3’ direction. This
strand is referred to as the leading
strand.
Leading strand
23

24. Semi-continuous replication
Okazaki fragment
3'
5'
24

25. • Many DNA fragments are synthesized
sequentially on the DNA template
strand having the 5´- end. These DNA
fragments are called Okazaki
fragments. They are 1000 – 2000 nt
long for prokaryotes and 100-150 nt
long for eukaryotes.
• The daughter strand consisting of
Okazaki fragments is called the
lagging strand.
Okazaki fragments
25

26. Continuous synthesis of the leading
strand and discontinuous synthesis of
the lagging strand represent a unique
feature of DNA replication. It is
referred to as the semi-continuous
replication.
Semi-continuous replication
26

27. Section 2
Enzymology
of DNA Replication

28. Enzymes and protein factors
protein Size function
Dna A protein 50,000 recognize origin
Dna B protein 300,000 open dsDNA
Dna C protein 29,000 assist Dna B binding
DNA pol Elongate the DNA
strands
Dna G protein 60,000 synthesize RNA primer
SSB 75,600 single-strand binding
DNA topoisomerase 400,000 release supercoil
constraint
28

29. • The first DNA-
dependent DNA
polymerase (short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
§2.1 DNA Polymerase
DNA-pol of prokaryotes
29

30. • Later, DNA-pol II and DNA-pol III were
identified in experiments using
mutated E.coli cell line.
• All of them possess the following
biological activity.
1. 53 polymerizing
2. exonuclease
30

31. DNA-pol I
• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
31

32. DNA-pol II
• Temporary functional when DNA-pol I
and DNA-pol III are not functional
• Still capable for doing synthesis on
the damaged template
• Participating in DNA repairing
32

33. DNA-pol III
• A heterodimer enzyme composed of
ten different subunits
• Having the highest polymerization
activity (105 nt/min)
• The true enzyme responsible for the
elongation process
33

34. §2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers
using free NTPs as the substrate and
the ssDNA as the template.
• Primers are short RNA fragments of a
several decades of nucleotides long.
34

35. §2.3 Helicase
• Also referred to as DnaB.
• It opens the double strand DNA with
consuming ATP.
• The opening process with the
assistance of DnaA and DnaC
35

36. §2.4 SSB protein
• Stand for single strand DNA binding
protein
• SSB protein maintains the DNA
template in the single strand form in
order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
36

37. §2.5 Topoisomerase
• Opening the dsDNA will create
supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
37

38. 38

39. • Also called -protein in prokaryotes.
• It cuts a phospho-diester bond on one
DNA strand, rotates the broken DNA
freely around the other strand to relax
the constraint, and reseals the cut.
Topoisomerase I (topo I)
39

40. • It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both
strands of dsDNA, releases the
supercoil constraint, and reforms the
phosphoester bonds.
• It can change dsDNA into the
negative supercoil state with
consumption of ATP.
Topoisomerase II (topo II)
40

41. • Connect two adjacent ssDNA strands
by joining the 3´-OH of one DNA
strand to the 5´-P of another DNA
strand.
• Sealing the nick in the process of
replication, repairing, recombination,
and splicing.
41
§2.6 DNA Ligase

42. §2.7 Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use two mechanisms to
ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
42

43. • DNA-pol I has the function to correct
the mismatched nucleotides.
• It identifies the mismatched
nucleotide, removes it using the 3´-
5´ exo-nuclease activity, add a correct
base, and continues the replication.
Proofreading and correction
43

44. Section 3
DNA Replication
Process

45. DNA
helicas
e
DNA helicases unwind the double helix
Replication in details

46. The formation of replication fork and
SSBs bind to stabilise ssDNA

47. Topoisomerases remove positive supercoils .

48. DNA replication requires a RNA primer
Primase make short RNA primers using
ssDNA as template
DNA primase is activated by interacting
with the DNA helicase
PRIMASE
SHORT RNA
PRIMERS

49. DNA polymerases
catalyzes DNA
synthesis

50. Sliding DNA
clamps increases
processivity
SLIDING DNA
CLAMPS
Processivity : the ability of
an enzyme to catalyze many
reactions before releasing
its substrate

51. RNAse degrades RNA
base paired with DNA
Removal of RNA primers
leaves gaps
DNA polymerase fill the
gaps
DNA ligase repairs the
remaining nicks

52. • Initiation: recognize the starting point,
separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing
strand, form phosphoester bonds,
correct the mismatch bases, extending
the DNA strand, …
• Termination: stop the replication
Sequential actions
52